Systems and methods for the amplification of dna

ABSTRACT

A system for amplifying nucleic acids is disclosed which, in one embodiment, includes a fluidic device having a sample channel and a heat exchange channel disposed sufficiently close to the sample channel such that a heat exchange fluid in the heat exchange channel can cause a sample in the sample channel to gain or lose heat at desired levels. In one illustrative embodiment, the system further includes three reservoirs coupled to the heat exchange channel and a temperature control system configured to heat fluids stored in the respective reservoirs at different temperatures. One or more pumps and a controller are configured to cause fluid stored in the reservoirs to enter and flow through the heat exchange channel at different times.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.12/233,194, filed on Sep. 18, 2008, the disclosure of which isincorporated herein by reference in its entirety.

BACKGROUND

Field of the Invention

The present invention relates to systems and methods for amplifyingnucleic acids. In some embodiments, the invention relates tomicrofluidic PCR analysis systems using microfluidic temperaturecontrolled channels.

Discussion of the Background

The amplification and detection of nucleic acids is central to medicine,forensic science, industrial processing, crop and animal breeding, andmany other fields. The ability to detect disease conditions (e.g.,cancer), infectious organisms (e.g., HIV), genetic lineage, geneticmarkers, and the like, is ubiquitous technology for disease diagnosisand prognosis, marker assisted selection, correct identification ofcrime scene features, the ability to propagate industrial organisms andmany other techniques. Determination of the integrity of a nucleic acidof interest can be relevant to the pathology of an infection or cancer.One of the most powerful and basic technologies to detect smallquantities of nucleic acids is to replicate some or all of a nucleicacid sequence many times, and then analyze the amplification products.PCR is perhaps the most well-known of a number of differentamplification techniques.

PCR is a powerful technique for amplifying short sections of DNA. WithPCR, one can quickly produce millions of copies of DNA starting from asingle template DNA molecule. PCR includes a three phase temperaturecycle of denaturation of DNA into single strands, annealing of primersto the denatured strands, and extension of the primers by a thermostableDNA polymerase enzyme. This cycle is repeated so that there are enoughcopies to be detected and analyzed. In principle, each cycle of PCRcould double the number of copies. In practice, the multiplicationachieved after each cycle is always less than 2. Furthermore, as PCRcycling continues, the buildup of amplified DNA products eventuallyceases as the concentrations of required reactants diminish. For generaldetails concerning PCR, see Sambrook and Russell, Molecular Cloning—ALaboratory Manual (3rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (2000); Current Protocols in Molecular Biology,F. M. Ausubel et al., eds., Current Protocols, a joint venture betweenGreene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(supplemented through 2005) and PCR Protocols A Guide to Methods andApplications, M. A. Innis et al., eds., Academic Press Inc. San Diego,Calif. (1990).

Real-time PCR refers to a growing set of techniques in which onemeasures the buildup of amplified DNA products as the reactionprogresses, typically once per PCR cycle. Monitoring the accumulation ofproducts over time allows one to determine the efficiency of thereaction, as well as to estimate the initial concentration of DNAtemplate molecules. For general details concerning real-time PCR seeReal-Time PCR: An Essential Guide, K. Edwards et al., eds., HorizonBioscience, Norwich, U.K. (2004).

Several different real-time detection chemistries now exist to indicatethe presence of amplified DNA. Most of these depend upon fluorescenceindicators that change properties as a result of the PCR process. Amongthese detection chemistries are DNA binding dyes (such as SYBR® Green)that increase fluorescence efficiency upon binding to double strandedDNA. Other real-time detection chemistries utilize Forster resonanceenergy transfer (FRET), a phenomenon by which the fluorescenceefficiency of a dye is strongly dependent on its proximity to anotherlight absorbing moiety or quencher. These dyes and quenchers aretypically attached to a DNA sequence-specific probe or primer. Among theFRET-based detection chemistries are hydrolysis probes and conformationprobes. Hydrolysis probes (such as the TaqMan® probe) use the polymeraseenzyme to cleave a reporter dye molecule from a quencher dye moleculeattached to an oligonucleotide probe. Conformation probes (such asmolecular beacons) utilize a dye attached to an oligonucleotide, whosefluorescence emission changes upon the conformational change of theoligonucleotide hybridizing to the target DNA.

A number of commercial instruments exist that perform real-time PCR.Examples of available instruments include the Applied Biosystems PRISM7500, the Bio-Rad iCylcer, and the Roche Diagnostics LightCycler 2.0.The sample containers for these instruments are closed tubes whichtypically require at least a 10 μl volume of sample solution.

More recently, a number of high throughput approaches to performing PCRand other amplification reactions have been developed, e.g., involvingamplification reactions in microfluidic devices, as well as methods fordetecting and analyzing amplified nucleic acids in or on the devices.Thermal cycling of the sample for amplification is usually accomplishedin one of two methods. In the first method, the sample solution isloaded into the device and the temperature is cycled in time, much likea conventional PCR instrument. In the second method, the sample solutionis pumped continuously through spatially varying temperature zones.

To have good yield of a target product, one has to control the sampletemperature at different levels very accurately. And to reduce theprocess time, one has to heat up or cool down the sample to desiredtemperature very quickly.

One specific approach for regulating temperature within the devices isto employ external temperature control sources. Examples of such sourcesinclude, but are not limited to, heating blocks and water baths. Anotheroption is to utilize a heating element such as a resistive heater thatcan be adjusted to a particular temperature. Another temperaturecontroller includes Peltier controllers (e.g., INB Productsthermoelectric module model INB-2-(11-4)1.5). This controller can beutilized to achieve effective thermal cycling or to maintain isothermalincubations at any particular temperature.

In some devices and applications, heat exchangers can also be utilizedin conjunction with one of the temperature control sources to regulatetemperature. Such heat exchangers typically are made from variousthermally conductive materials (e.g., various metals and ceramicmaterials) and are designed to present a relatively large externalsurface area to the adjacent region. Often this is accomplished byincorporating fins, spines, ribs and other related structures into theheat exchanger. Other structures include coils and sintered structures.In certain devices, heat exchangers such as these are incorporated intoa holding space, chamber or detection area.

Conventional heat exchangers that can be utilized in certainapplications are discussed, for example, in U.S. Pat. No. 6,171,850which discloses a reaction receptacle that includes a plurality ofreservoirs disposed in the surface of a substrate. Additional methods oftemperature control for microfluidic systems are known which include,for example: a thermal cycling system using the circulation oftemperature controlled water to the underside of a microtiter plate(U.S. Pat. No. 5,508,197); a thermal cycling system using infraredheating and air cooling (U.S. Pat. No. 6,413,766); a microfluidic chipwhere flow travels through several static temperature zones (U.S. Pat.No. 6,960,437); the use of exothermic and endothermic materials to heatup and cool down the PCR samples (U.S. patent application publicationUS2005/012982).

In conventional systems temperature accuracy and thermal cycling speedsare issues to be resolved. For example, the accuracy of the temperatureof any bath used to heat a microchannel and the bath's subsequentconduction of heat to the microchannel is important in that certainstages of PCR processing take place at well-defined temperatures. Thethermal cycling speed refers to the time between stabilization from onetemperature to another in a heating cycle. For example in the PCRprocess, the thermal cycling speed refers to the time to shift from 95°C. to 55° C. to 72° C. The faster the thermal cycling speeds and themore accurate the temperature stabilization, the more efficient PCRprocesses can be performed.

There is a need for improved systems and methods for amplifying nucleicacids and for systems and methods for microfluidic thermal control.

SUMMARY

The present invention provides improved systems and methods foramplifying nucleic acids and systems and methods for microfluidictemperature control.

A method according to some embodiments of the invention includes:causing a sample of a test solution containing PCR reagents to movethrough a sample channel of a fluidic device and while the sample ismoving through at least a section of the sample channel: (1) for a firstperiod of time, causing a first heat exchange fluid stored in a firstcontainer and regulated at a first temperature while stored in the firstcontainer to exit the first container and move through a heat exchangechannel of the fluidic device after exiting the first container; (2) fora second period of time, causing a second heat exchange fluid stored ina second container and regulated at a second temperature while stored inthe second container to exit the second container and move through theheat exchange channel after exiting the second container; and (3) for athird period of time, causing a third heat exchange fluid stored in athird container and regulated at a third temperature while stored in thethird container to exit the third container and move through the heatexchange channel after exiting the third container. Steps (1)-(3) arepreferably repeated at least several times. Also, it is preferred thatthe first period of time is different than the second period of time,which is different than the third period of time, although there may besome overlap between the time periods. It is also preferred that thefirst temperature is different than the second temperature, which isdifferent than the third temperature.

In some embodiments, the method may further include causing the firstheat exchange fluid to enter the third container after exiting the heatexchange channel, causing the second heat exchange fluid to enter thefirst container after exiting the heat exchange channel, and causing thethird heat exchange fluid to enter the second container after exitingthe heat exchange channel. The heat exchange fluids may be a gas, aliquid or a gas and liquid mixture. For example, the heat exchangefluids may include water and/or compressed air with pressure from 1 to200 psia.

The heat exchange and sample channels may each have a dimension lessthan 2000 micrometers. For example, the heat exchange channel may have awidth between about 20 and 2000 micrometers and a depth between about 20and 2000 micrometers. The containers may have a volume of less than 2000ml. For example, the containers may have a volume from 10 to 1000 ml.

A system according to an embodiment of the invention includes: a fluidicdevice comprising a sample channel and a heat exchange channelsufficiently close to the sample channel such that a heat exchange fluidin the heat exchange channel can cause a sample in the sample channel toappreciably gain or lose heat; a first reservoir having an output portcoupled to an input of the heat exchange channel and having an inputport coupled to an output of the heat exchange channel through a firstreturn valve, the first reservoir storing a first heat exchange fluid; asecond reservoir having an output port coupled to the input of the heatexchange channel and having an input port coupled to the output of theheat exchange channel through a second return valve, the secondreservoir storing a second heat exchange fluid; a third reservoir havingan output port coupled to the input of the heat exchange channel andhaving an input port coupled to the output of the heat exchange channelthrough a third return valve, the third reservoir storing a third heatexchange fluid; a temperature control system; one or more pumps; and acontroller.

The temperature control system may be configured to: (a) regulate theheat exchange fluid stored in the first reservoir at a firsttemperature, (b) regulate the heat exchange fluid stored in the secondreservoir at a second temperature, and (c) regulate the heat exchangefluid stored in the third reservoir at a third temperature.

The controller may be configured to operate the valves and the one ormore pumps such that: (a) for a first period of time, the first heatexchange fluid stored in the first reservoir enters the heat exchangechannel, but the second and third heat exchange fluids stored in thesecond and third reservoirs, respectively, do not enter the heatexchange channel; (b) for a second period of time, the second heatexchange fluid stored in the second reservoir enters the heat exchangechannel, but the first and third heat exchange fluids stored in thefirst and third reservoirs, respectively, do not enter the heat exchangechannel; and (c) for a third period of time, the third heat exchangefluid stored in the third reservoir enters the heat exchange channel,but the first and second heat exchange fluids stored in the first andsecond reservoirs, respectively, do not enter the heat exchange channel.

In other embodiments, a thermal exchange system for microfluidic systemsincludes at least one heat exchange channel, wherein the at least oneheat exchange channel is configured to carry a heat exchange fluid,wherein the heat exchange channel is configured to exchange heat with aportion of a sample channel, wherein the sample channel is configured tocarry a genomic sample in a buffer. The system further includes at leasttwo reservoir tanks, a first reservoir tank and a second reservoir tank,wherein the first reservoir tank is configured to include a first heatexchange fluid at a first temperature, and the second reservoir tank isconfigured to include a second heat exchange fluid at a secondtemperature, wherein either the first or the second heat exchange fluidscan be directed into the at least one heat exchange channel. In otheraspects of this system, three reservoirs are included wherein the thirdreservoir includes a third heat exchange fluid at a third temperature.

The thermal exchange system according to one embodiment is furthercharacterized in that the first heat exchange fluid is flowing throughthe at least one heat exchange channel and the portion of the samplechannel is heated to about 95 degrees Celsius, the second heat exchangefluid is flowing through the at least one heat exchange channel and theportion of the sample channel is heated to about 55 degrees Celsius, andthe third heat exchange fluid is flowing through the at least one heatexchange channel and the portion of the sample channel is heated toabout 72 degrees Celsius.

In some embodiments, the thermal exchange system has at least one heatexchange channel that is substantially parallel to the sample channel.In other embodiments, the thermal exchange system has at least one heatexchange channel that is substantially perpendicular to the samplechannel. In still other embodiments, the thermal exchange system has atleast one heat exchange channel that is configured to exchange heat withsubstantially one side of the sample channel. In yet other embodiments,the thermal exchange system has at least one heat exchange channel thatis configured to exchange heat with substantially two sides or threesides of the sample channel.

The above and other embodiments of the present invention are describedbelow with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated herein and form partof the specification, illustrate various embodiments of the presentinvention. In the drawings, like reference numbers indicate identical orfunctionally similar elements.

FIG. 1 is a block diagram illustrating a system according to embodimentsof the invention.

FIGS. 2A-2B illustrate a temperature control system according to someembodiments of the invention.

FIGS. 3A-3C illustrate various configurations of a heat exchange channelaccording to embodiments of the invention.

FIG. 4 illustrates a process according to some embodiments of theinvention.

FIGS. 5A-5B illustrate portions of processes according to someembodiments of the invention.

FIG. 5C illustrates a temperature profile that can result from the useof a temperature control system according to at least one exemplaryembodiment.

FIG. 6 illustrates a temperature control system according to otherembodiments of the invention.

FIG. 7 illustrates a portion of a process according to some embodimentsof the invention.

FIG. 8 illustrates a temperature control system in accordance with otherembodiments of the invention.

FIG. 9 illustrates a temperature control system in accordance with stillother embodiments of the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

FIG. 1 illustrates a functional block diagram of a system 100 for theamplification of DNA according to some embodiments of the invention. Asillustrated in FIG. 1, system 100 may include a microfluidic device 102.Microfluidic device 102 may include one or more microfluidic channels104. In the example shown, device 102 includes two microfluidicchannels, channel 104 a and channel 104 b. Although only two channelsare shown in the exemplary embodiment, it is contemplated that device102 may have fewer than two or more than two channels. For example, insome embodiments, device 102 includes eight channels 104.

Device 102 may include two DNA processing zones, a DNA amplificationzone 131 (a.k.a., PCR zone 131) and a DNA melting zone 132. A DNA sampletraveling through the PCR zone 131 may undergo PCR, and a DNA samplepassing through melt zone 132 may undergo high resolution thermalmelting. As illustrated in FIG. 1, PCR zone 131 includes a first portionof channels 104 and melt zone 132 includes a second portion of channels104, which is down stream from the first portion.

In order to achieve PCR for a DNA sample flowing through the PCR zone131, the temperature of the sample must be cycled, as is well known inthe art. Accordingly, in some embodiments, system 100 includes atemperature control apparatus 120. The temperature control apparatus 120may include a temperature sensor, a heater/cooler, and a temperaturecontroller. In some embodiments, temperature controller 120 isinterfaced with main controller 130 so that main controller 130 cancontrol the temperature of the samples flowing through the PCR zone andthe melting zone.

To monitor the PCR process and the thermal melting process that occur inPCR zone 131 and melt zone 132, respectively, system 100 may include animaging system 118. Imaging system 118 may include an excitation source,a detector, a controller, and an image storage unit.

Further features of system 100 are described in U.S. patent applicationSer. No. 11/770,869, which is incorporated herein by this reference.

FIGS. 2A and 2B further illustrate a temperature control apparatus 120in accordance with some embodiments of the invention. FIG. 2Aillustrates a single heat exchange channel 210 and a single samplechannel 104 (although, as discussed above, the invention is not limitedto a single heat exchange channel 210 and/or sample channel 104). Theheat exchange channel 210 is configured to carry a heat exchange fluidand configured to exchange heat with a portion 230 of a sample channel220. The sample channel 220 can be configured to carry a bolus 240 ofgenomic sample material, which experiences temperature variation due tothe heat exchange through the portion 230. In the one exemplaryembodiment, temperature variations are performed temporally by varyingthe temperature of the heat exchange fluid temporally or by switching toa different heat exchange fluid at a particular time that has adifferent temperature.

At least one exemplary embodiment includes multiple reservoirs ofvarious heat exchange fluids at various temperatures. For example, FIG.2B illustrates a reservoir system for storing and directing heatexchange fluids through the heat exchange channel 210. FIG. 2Billustrates three fluid containers (a.k.a., reservoirs) T1, T2, and T3,each of which stores a fluid (e.g., a gas, a liquid or a gas and liquidmixture) and pump 290 coupled to each container for pumping fluid out ofthe containers and into a heat exchange channel 210 formed in chip 102.In one embodiment, the first heat exchange fluid is identical with thesecond heat exchange fluid, which is identical with the third heatexchange fluid, and the first heat exchange fluid comprises a gas and/ora liquid. In other embodiments, the heat exchange fluids comprise waterand/or compressed air with pressure from 1 to 200 psia. In still otherembodiments, the heat exchanges fluids can be different from oneanother. In one non-limiting example, the first heat exchange fluid is agas, the second heat exchange fluid is a liquid and the third heatexchange fluid is gas and liquid mixture..

Each container T1-T3 includes an output port that is coupled to an inputof the heat exchange channel through a forward valve. For example, theoutput port of T1 is coupled to the heat exchange channel throughforward valve V1F, the output port of T2 is coupled to the heat exchangechannel through forward valve V2F, and the output port of T3 is coupledto the heat exchange channel through forward valve V3F.

Each container T1-T3 also includes an input port that is coupled to anoutput of the heat exchange channel through a return valve. For example,the input port of T1 is coupled to the heat exchange channel throughreturn valve V1R, the input port of T2 is coupled to the heat exchangechannel through return valve V2R, and the input port of T3 is coupled tothe heat exchange channel through return valve V3R.

As further illustrated, temperature control apparatus 120 may include atemperature control system that includes one or more temperaturescontrollers. For example, in the illustrated embodiment of FIG. 2B,temperature control apparatus 120 includes a temperature controller C1for regulating the temperature of the fluid stored in T1 at a firsttemperature (e.g., C1 attempts to maintain the temperature of the fluidin T1 at, or close to, a predetermined temperature), a temperaturecontroller C2 for regulating the temperature of the fluid stored in T2at a second temperature, and a temperature controller C3 for regulatingthe temperature of the fluid stored in T3 at a third temperature. Eachof C1, C2 and C3 may include, a sensor for sensing temperature,heating/cooling elements, and computerized controllers for controllingthe heating/cooling elements based on output from a sensor.

Referring now to FIGS. 3A-C, cross-sectional, end views of chip 102 areshown and serve to illustrate various different embodiments of heatexchange channel 304 and to illustrate the relationship between a samplechannel 104, which carries a sample 302, and heat exchange channel 304.Sample 302 may include a solution that contains, among other things, apiece of DNA, DNA polymerase, and a primer.

As illustrated in FIGS. 3A-C, heat exchange channel 304 may only runalong one side of channel 104 (see FIG. 3A), heat exchange channel 304may be generally L shaped and run along two sides of channel 104 (seeFIG. 3B), and heat exchange channel 304 may be generally U shaped andrun along three side of channel 104 (see FIG. 3C). In some embodiments,channel 304 may have a width between about 10 and 3000 micrometers (morepreferably between about 20 and 2000 micrometers) and a depth betweenabout 10 and 3000 micrometers (more preferably between about 20 and 2000micrometers).

Referring now to FIG. 4, a flow chart illustrates a process 400according to some embodiments of the invention. Process 400 may begin instep 402, where a fluid is stored in a first container (e.g., containerT1). In step 404, the temperature of the fluid in the first container isregulated at a first temperature (e.g., at least about 80 degreesCelsius). In step 406, a fluid is stored in a second container (e.g.,container T2). In step 408, the temperature of the fluid in the secondcontainer is regulated at a second temperature (e.g., a temperature notmore than about 60 degrees Celsius). In step 410, a fluid is stored in athird container (e.g., container T3). In step 412, the temperature ofthe fluid in the third container is regulated at a third temperature(e.g., a temperature between about 60 and 80 degrees Celsius). In step414, a sample (e.g., sample 302) is caused to flow though sample channel104. While the sample is flowing through channel 104, steps 416-420 canbe performed.

In step 416, the fluid stored in the first container is caused to flowthrough heat exchange channel 304 for a first amount of time. Next, instep 418, the fluid stored in the second container is caused to flowthrough heat exchange channel 304 for a second amount of time. Next, instep 420, the fluid stored in the third container is caused to flowthrough heat exchange channel 304 for a third amount of time. After step420, steps 416-420 may be repeated a number of times. The first amountof time may be different than the second amount of time, which may bedifferent than the third amount of time.

In one exemplary, non-limiting embodiment, the fluid stored in the firstcontainer (e.g. water) can be heated to a temperature of approximately97 degrees Celsius so that the sample material can be heated to atemperature of approximately 95 degrees Celsius. The fluid stored in thesecond container (e.g. water) can be maintained at a temperature ofapproximately 53 degrees Celsius so that the sample material can becooled to a temperature of approximately 55 degrees Celsius. The fluidstored in the third container (e.g. water) can be heated to atemperature of approximately 74 degrees Celsius so that the samplematerial can be heated to a temperature of approximately 72 degreesCelsius. Also in this exemplary embodiment, the fluid stored in thefirst container is caused to flow through heat exchange channel 304 fora first amount of time that can be, for example, approximately 0.3 to 2seconds and preferably approximately 0.5 seconds. The fluid stored inthe second container is caused to flow through heat exchange channel 304for a second amount of time that can be, for example, approximately 1 to5 seconds and preferably approximately 2 seconds. The fluid stored inthe third container is caused to flow through heat exchange channel 304for a third amount of time that can be, for example, approximately 1 to10 seconds and preferably approximately 5 seconds. Of course, the fluidstored in the containers can be heated or cooled to differenttemperatures and the time periods during which the fluid flows throughthe heat exchange channel can be decreased or increased depending on therequirements for a given amplification reaction.

Referring now to FIG. 5A, steps 416-420 are further illustratedaccording to some embodiments where process 400 is implemented using theapparatus shown in FIGS. 2A-B. As shown in FIG. 5A, step 416 may includeopening valve V1F, opening valve V3R and closing the other valves (V2F,V3F, V1R, and V2R), step 418 may include opening valve V2F, openingvalve V1R and closing the other valves (V1F, V3F, V2R, and V3R), step420 may include opening valve V3F, opening valve V2R and closing theother valves (V1F, V2F, V1R, and V3R). Preferably, while all the steps416-420 are being performed, pump 290 is activated, thereby causing thefluids to flow out of a container and back into a container. Thecontainer from which the fluid flows and to which the fluid returns, ofcourse, depends on the valves that are open at the time. For example,when step 416 is performed in accordance with the flow shown in FIG. 5A,fluid will flow out of container T1 and into container T3. Directing thefluid flow out of container T1 and into container T3, in this particularembodiment, is one exemplary way to allow more time for the fluid toreach the desired temperature level, which can increase the temperatureaccuracy and efficiency of the temperature cycling process.

Referring now to FIG. 5B, steps 416-420 are further illustratedaccording to another embodiment where process 400 is implemented usingthe apparatus shown in FIGS. 2A-B. As shown in FIG. 5B, step 416 mayinclude opening valve V1F, opening valve V1R and closing the othervalves (V2F, V3F, V2R, and V3R), step 418 may include opening valve V2F,opening valve V2R and closing the other valves (V1F, V3F, V1R, and V3R),step 420 may include opening valve V3F, opening valve V3R and closingthe other valves (V1F, V2F, V1R, and V2R). Preferably, while all thesteps 416-420 are being performed, pump 290 is activated, therebycausing the fluids to flow out of a container and back into a container.In this example, when step 416 is performed in accordance with the flowshown in FIG. 5B, fluid will flow, for example, out of container T1 andback into container T1.

In another embodiment, one or more of the containers T1-T3 areconstructed to have an internal bladder or baffle that separates theinternal portion of the container into a first chamber and a secondchamber, and wherein the first and second chambers are in fluidcommunication with one another by, for example, a controllable valve. Inthis embodiment, fluid can be controllably released from one chamber ofthe container (e.g. T1) through a forward valve (e.g. V1F) and can becontrollably caused to flow back into the other chamber of the containerthrough the return valve (e.g. V1R). As stated above, fluid also cancontrollably flow between the first chamber and the second chamber of acontainer through, for example, a controllable valve in the bladder orbaffle separating the chambers. This embodiment may be useful, forexample, in an embodiment where fluid flows out one container and backinto the same container before fluid flows out of, or into, anothercontainer, as discussed in connection with the process illustrated inFIG. 5B. This embodiment also may be useful in connection with otherembodiments where different fluids are used in the containers such as,for example, when a gas is used in container T1, a liquid is used incontainer T2 and a mixture of gas and liquid is used in container T3.

FIG. 5C illustrates an example of a temperature versus time plot of thetemperature experienced by bolus 240 as it traverses through the samplechannel 220 as various heat exchange fluids flow through the heatexchange channel 210 at various times, in accordance with at least oneexemplary embodiment of the present invention.

Referring now to FIG. 6, an apparatus 120 is illustrated according toanother embodiment. The embodiment shown in FIG. 6 is similar to thatshown in FIGS. 2A-B, with the exception that the forward valves V1F,V2F, and V3F are replaced with pumps 602, 604 and 606, respectively, andpump 290 is not present. The operation of the apparatus 120 inaccordance with this embodiment is discussed below.

Referring now to FIG. 7, steps 416-420 are further illustrated accordingto some embodiments where process 400 is implemented using the apparatusshown in FIG. 6. As shown in FIG. 7, step 416 may include, activatingonly pump 602, opening valve V3R and closing the other return valves(V1R and V2R), step 418 may include activating only pump 604, openingvalve V1R and closing the other return valves (V3R and V2R), step 420may include activating only pump 606, opening valve V2R and closing theother return valves (V1R and V3R). The container from which the fluidflows and to which the fluid returns, of course, depends on the valvesthat are open at the time and the pump that is activated. For example,when step 418 is performed in accordance with the flow shown in FIG. 7,fluid will flow out of container T2 and into container T1. The apparatusof FIG. 6 also can be controlled such that fluid will flow out of onecontainer and back into the same container, as discussed above.

FIG. 8 illustrates a thermal exchange system 800 in accordance withanother exemplary embodiment. The thermal exchange system 800 isdirected to a thermal exchange system that includes a plurality of heatexchange channels (e.g., 810 a-c), each configured to carry a heatexchange fluid, where each heat exchange fluid preferably is at adifferent temperature. The plurality of heat exchange channels (810 a-c)can be configured to lie substantially perpendicular (orthogonal)(although the invention is not limited to an orthogonal orientation) toa sample channel 820 and configured to exchange heat with a portion(e.g., 830 a-c) of the sample channel. In this exemplary embodiment, abolus 840 of genomic material traveling along the sample channel 820experiences temperature change associated with heat exchanged in fromportions of heat exchange channels 830 a-c, as discussed above inconnection with other embodiments.

In the embodiment of FIG. 8, heat exchange channel 810 a is in fluidcommunication with at least container T1, heat exchange channel 810 b isin fluid communication with at least container T2, and heat exchangechannel 810 c is in fluid communication with at least container T3. Inone aspect of this embodiment, fluid is caused to flow from containersT1-T3 and through heat exchange channels 810 a-c through one or morepumps and is caused to return to the containers through one or morereturn valves, as disclosed herein. The container from which the fluidflows and to which the fluid returns, of course, depends on the valvesthat are open at the time and the pump that is activated.

FIG. 9 illustrates a thermal exchange system 900 in accordance withanother exemplary embodiment, which includes a curved sample channel920, that directs a bolus 940 of genomic material back and forth(905A-C) near a plurality of heat exchange channels (910 a-c), eachconfigured to carry a heat exchange fluid, where each heat exchangefluid preferably is at a different temperature. In this exemplaryembodiment, a bolus 940 of genomic material traveling along the curvedsample channel 920 experiences temperature change associated with heatexchanged with portions of heat exchange channels 910 a-c, as discussedabove in connection with other embodiments. In this embodiment, thefluid flows from the containers T1-T3 through heat exchange channels 910a-c, for example, in the same manner described above in connection withthe FIG. 8 embodiments and other embodiments described herein.

While various embodiments/variations of the present invention have beendescribed above, it should be understood that they have been presentedby way of example only, and not limitation. Thus, the breadth and scopeof the present invention should not be limited by any of theabove-described exemplary embodiments. Further, unless stated, none ofthe above embodiments are mutually exclusive. Thus, the presentinvention may include any combinations and/or integrations of thefeatures of the various embodiments.

Additionally, while the processes described above and illustrated in thedrawings are shown as a sequence of steps, this was done solely for thesake of illustration. Accordingly, it is contemplated that some stepsmay be added, some steps may be omitted, and the order of the steps maybe re-arranged.

What is claimed is:
 1. A method of amplifying DNA using a systemcomprising a fluidic device having a sample channel and a heat exchangechannel that is used to heat and/or cool a sample in the sample channel,the method comprising: causing a sample of a test solution containingPCR reagents to move through the sample channel of the fluidic device;and while the sample is moving through at least a section of the samplechannel: (1) for a first period of time, causing a first heat exchangefluid stored in a first container and regulated at a first temperaturewhile stored in the first container to: (1) exit the first container and(2) move through the heat exchange channel after exiting the firstcontainer; (2) for a second period of time, causing a second heatexchange fluid stored in a second container and regulated at a secondtemperature while stored in the second container to: (1) exit the secondcontainer and (2) move through the heat exchange channel after exitingthe second container; and (3) for a third period of time, causing athird heat exchange fluid stored in a third container and regulated at athird temperature while stored in the third container to: (1) exit thethird container and (2) move through the heat exchange channel afterexiting the third container, wherein the first period of time may bedifferent than the second period of time, which may be different thanthe third period of time, and the first temperature is different thanthe second temperature, which is different than the third temperature.2. The method of claim 1, further comprising causing the first heatexchange fluid to enter the third container after exiting the heatexchange channel.
 3. The method of claim 2, further comprising causingthe second heat exchange fluid to enter the first container afterexiting the heat exchange channel.
 4. The method of claim 3, furthercomprising causing the third heat exchange fluid to enter the secondcontainer after exiting the heat exchange channel.
 5. The method ofclaim 1, further comprising causing the first heat exchange fluid toenter the first container after exiting the heat exchange channel. 6.The method of claim 5, further comprising causing the second heatexchange fluid to enter the second container after exiting the heatexchange channel.
 7. The method of claim 6, further comprising causingthe third heat exchange fluid to enter the third container after exitingthe heat exchange channel.
 8. The method of claim 1, wherein the firsttemperature is a temperature such that when the first heat exchangefluid moves through the heat exchange channel said fluid heats a samplein the sample channel to a temperature over 80 degrees Celsius, thesecond temperature is a temperature such that when the second heatexchange fluid moves through the heat exchange channel said fluid coolsa sample in the sample channel to a temperature under about 60 degreesCelsius, and the third temperature is a temperature such that when thethird heat exchange fluid moves through the heat exchange channel saidfluid heats a sample in the sample channel to a temperature between 60and 80 degrees Celsius.
 9. The method of claim 1, wherein at least aportion of the heat exchange channel is beneath the sample channel andparallel with the sample channel.
 10. The method of claim 1, wherein atleast one dimension of the heat exchange channel and the sample channelis less than about 3000 micrometers.
 11. The method of claim 10, whereinthe heat exchange channel has a width between about 20 and 2000micrometers and a depth between about 20 and 2000 micrometers.
 12. Themethod of claim 1, wherein said first heat exchange fluid is identicalwith the second heat exchange fluid, which is identical with the thirdheat exchange fluid, and the first heat exchange fluid comprises a gasand/or a liquid.
 13. The method of claim 1, wherein said heat exchangefluids comprise water and/or compressed air with pressure from 1 to 200psia.
 14. The method of claim 1, wherein said first heat exchange fluidis different than the second heat exchange fluid, which can be the sameor different than the third heat exchange fluid.
 15. A method of thermalexchange in a microfluidic chip comprising: directing a first heatexchange fluid at a first temperature through a heat exchange channelfor a first period of time, wherein the heat exchange channel isconfigured to exchange heat with a portion of a sample channel, whereinat least one dimension of the heat exchange channel and the samplechannel are less than 1000 micrometers; directing a second heat exchangefluid at a second temperature through the heat exchange channel for asecond period of time; and directing a third heat exchange fluid at athird temperature through a heat exchange channel for a third period oftime.